The present invention relates to a chloroperoxidase enzyme preparation, and in particular, to a fungal chloroperoxidase enzyme preparation having a pH optimum above pH 5.0 and the ability to oxidize chloride, bromide, and iodide ions.
There has been considerable interest in recent years in enzymatic halogenation. U.S. Pat. Nos. 4,247,641 and 4,284,723 describe the use of haloperoxidase enzymes to produce epoxides from alkenes. Other recent research activity in this field has involved the use of haloperoxidase enzymes to produce halogenated ketones from alkynes, to produce alpha, gamma-halohydrins from cyclopropanes, and to produce dihalogenated products from alkenes and alkynes. The term haloperoxidase is used herein to include chloroperoxidases, bromoperoxidases, and iodoperoxidases. A chloroperoxidase, as that term is used herein, is an enzyme capable of oxidizing chloride, bromide, or iodide ions with the consumption of H.sub.2 O.sub.2. A bromoperoxidase can oxidize iodide and bromide, but not chloride ions, and an iodoperoxidase can oxidize iodide ions only, both of the latter enzymes requiring H.sub.2 O.sub.2 as a substrate.
Haloperoxidase enzymes which are known in the prior art and which have been used in halogenation-reaction studies include chloroperoxidase derived from the fungus Caldariomyces fumago, bromoperoxidase from algae, lactoperoxidase from milk, thyroid peroxidase from the thyroid, myeloperoxidase from leukocytes and horseradish peroxidase from horseradish. These enzymes are described generally in the Morrison, et al., Ann. Rev. Biochem., 45, 861 (1976).
Many industrial applications of halogenation involve chloride ions. The conversion of propylene to propylene oxide via chlorohydrin, described in Weissermel et al., in Industrial Organic Chemistry, 128, 260, (1978) is one example. Therefore, it is often advantageous that the haloperoxidase enzyme used in industrial halogenation be able to oxidize chloride ions.
Of the several known haloperoxidases which are mentioned above, only myeloperoxidase derived from leukocytes and chloroperoxidase from C. fumago are able to utilize chloride ions. A limitation of the C. fumago chloroperoxidase, in industrial applications, is that the enzyme has a pH optimum of around 3, and has relatively low activity and stability above pH 7.0. Thus, the enzyme preparation obtained by fermentation must first be acidified, and after product formation, the acid must be neutralized by the addition of base for product recovery or further conversion. The two pH adjustments significantly increase the cost of the halogenation reaction. Myeloperoxidase from leukocytes operates in the desired neutral pH range, but the enzyme is not readily obtained in quantities suitable for industrial use, as seen in U.S. Pat. No. 4,379,141, issued April 5, 1983.
It is one object of the present invention, therefore, to provide a haloperoxidase enzyme which combines important commercial advantages not found in any single haloperoxidase known heretofore.
A more specific object of the invention is to provide such an enzyme which is readily obtained by fermentation, has a pH optimum above about pH 5.0, and can utilize chloride ions.
It is still another object of the invention to provide a method of enzymatically halogenating a compound, at a selected pH between about 4 and 9, inclusive, using a fungal haloperoxidase derived from a or near-neutral pH. The structure classification of dematiaceous hyphomycetes follows that described in Ellis, M. B., Dematiaceous Hyphomycetes, pp. 7-23, Commonwealth Mycological Institute, Kew, Surrey, England, (1971) and in Ellis, M. B., More Dematiaceous Hyphomycetes, pp. 8-15, Commonwealth Mycological Institute, Kew, Surrey, England (1976). As outlined therein, the dematiaceous hyphomycetes can be divided into six groups based on characteristic morphology. The six groups are listed in Table I, the number of classified genera in each group being indicated in parentheses beside the group name.
TABLE I ______________________________________ No. of Genera Group No. ______________________________________ Thallic Non-meristematic (12) #1 Meristematic (2) #2 Blastic Basauxic (6) #3 Acroauxic Holoblastic (262) #4 Enteroblastic Tretic (36) #5 Phialidic (51) #6 ______________________________________
According to one aspect of the invention, a fungus selected from one of the six above-identified dematiaceous hyphomycete groups is cultured under conditions which are selected to optimize the measured haloperoxidase activity produced by the fungus. In a preferred protocol used in culturing the selected fungus, a small block from a sporulating fungal slant is transferred aseptically to a fungal agar seed plate and incubated at room temperature for a period typically between about 3 to 7 days. The fungal agar used in the fungus selected from the dematiaceous hyphomycetes.
A further object of the invention is to provide a method of oxidizing iodide ions enzymatically to molecular iodine.
Still another object of the invention is to provide a method of preparing such a haloperoxidase enzyme.
The invention includes a method for producing a halogenating enzyme which has a pH optimum above about pH 5.0, and which can oxidize chloride, bromide, or iodide ions in the presence of H.sub.2 O.sub.2. The method involves selecting a fungus from the dematiaceous hyphomycetes, and deriving from the selected fungus, an enzyme capable of brominating phenol red and halogenating monochlorodimedon in the presence of the appropriate halide and H.sub.2 O.sub.2 at a pH typically between about 7.0 and 8.5. Preferred organisms are selected from one of the genera including Alternaria, Curvularia, Drechslera, Ulocladium in the enteroblastic tretic group of dematiaceous hyphomycetes, and Botrytis in the acroauxic holoblastic group.
The enzyme is used in halogenating, at a selected pH between about 4 and 9, a compound preferably selected from the group consisting of alkenes, alkynes, cyclopropanes, beta-keto acids, cyclic beta-diketones and aromatic ring compounds. Preferred compounds include ethylene, propylene, monohalogenated analogues of these alkenes, and allyl alcohol.
These and other objects and features of the present invention will become more fully apparent from the following detailed description of the invention.